1. Field of Invention
This invention relates to novel nucleosides and dinucleoside dimers and derivatives of these compounds, including, L-deoxyribofuranosyl nucleoside phosphodiester dimers in which the sugar moiety of at least one of the nucleosides has an L-configuration. These compounds are highly effective in the treatment of various diseases, in particular the infection of the protozoan parasite, Plasmodium falciparum, the etiologic agent responsible for the most fatal form of malaria.
2. Prior Art
Modified nucleoside analogs are an important class of antineoplastic and antiviral drugs. The present application discloses novel compounds for of this type for use in the treatment of P. falciparum infection. Plasmodium falciparum is the etiologic agent responsible for the most fatal form of malaria, a disease which afflicts between 200 and 300 million people per year (all forms), including over one million childhood deaths. Additionally, greater than 40% of the world's population lives in areas in which malaria is at epidemic levels. Due to the extraordinary morbidity and mortality associated with malaria, malaria-related research has intensified during the past decade in a desperate search for an effective treatment. A safe and effective vaccine still does not exist. Instead, malaria victims must depend upon chemotherapy.
These chemotherapeutic agents can be classified into two groups: those that act post-translationally, and those that act by interfering with nucleic acid synthesis.
Most antimalarial drugs are in the first group, which means that they exert their therapeutic effect by interfering with the parasite cell's protein synthesis, and hence its metabolism (rather than its nucleic acid synthesis). Examples of drugs in this group include: the antifolate compounds (which inhibit dihyrdofolate reductase), and sulfonamide drugs (which inhibit dihydropteroate synthetase. Yet these drugs have serious drawbacks. Perhaps the most serious is that the protozoan responsible for malaria very quickly develops resistance to these drugs. The reason is that, since resistance occurs through adaptive mutations in successive generations of the parasite, one or two point mutation is often sufficient to confer resistance.
The second group of antimalarial compounds includes the nucleic acid intercalators such as acridines, phenanthrenes and quinolines. These intercalators partially mimic the biochemical activity of nucleic acids, and therefore are incorporated into the cell's nucleic acid (DNA and RNA), though once incorporated, do not allow further nucleic acid synthesis, hence their effectiveness. At the same time, these intercalators interfere with host nucleic acid synthesis as well, and thus give rise to toxic side effects. Because of the potential for toxic side effects, these drugs can quite often be given only in very small doses. Moreover, the protozoan responsible for malaria is known to develop "cross-resistance," which means that the parasite develops resistance to other classes of drugs even though it was exposed on a different class of drug.
Indeed, all of the currently known antimalarial drugs or drug candidates utilizing the delivery of cytotoxic pyrimidine or purine biosynthesis inhibitor to the malarial parasite are extremely toxic. Therefore, while drugs of this type--i.e., those that interfere with malarial parasite's nucleic acid synthesis-are effective, they lack selectivity. It is this latter parameter that must be maximized in the development of a safe and effective antimalarial drug. In other words, such a drug would target host tissues that are infected with the malarial protozoan yet leave the host tissue unchanged.
Recent advances in our understanding of the unusual biochemistry of parasite cells may prove valuable toward the design of an effective antimalarial therapy. One investigator (H. Ginsburg, Biochem. Pharmacol. 48, 1847-1856 (1994)) has observed that normal and parasite-infected erythrocytes exhibit significant differences with respect to purine and pyrimidine metabolism in single enzymes, as well as in whole branches of related pathways. The parasite satisfies all of its purine requirements through scavenger pathways; meanwhile, the host cell lacks the enzymes necessary to exploit this pathway, and so therefore must meet its pyrimidine requirements largely through de novo synthesis. Put another way, the parasite is more efficient than normal or host cells since it can synthesize the nucleic acid building blocks.
Other investigators (G. Beaton, D. Pellinqer, W. S. Marshall & M. H. Caruthers, In: Oligonucleotides and Analogues: A Practical Approach, F. Eckstein Ed., IRL Press, Oxford, 109-136 (1991)) have established that a malaria-infected erythrocyte is capable of effectively transporting the non-naturally occurring "L-nucleosides" (in contrast to the "D-nucleosides" which are the naturally occurring form) for use in nucleic acid synthesis. Yet, normal mammalian cells are nonpermeable to this class of compounds, which suggests that the L-nucleosides are non-toxic to normal mammalian or host cells. Thus, derivatives of these compounds may be used as highly selective drugs against parasite infection. The chemical modification of the L-nucleosides consists generally of modifying the nucleosides so that they are still recognized by the parasite's nucleic acid synthetic machinery, and therefore incorporated into a nucleic acid chain, but yet once this incorporation occurs, no further synthesis will take place.
Currently, there are no therapeutic compounds in use that are based on dimers of these nucleoside analogs. While dimers of the naturally occurring D-deoxyribofuranosyl nucleosides are well known, dimers in which one or both nucleosides are of the unnatural L-configuration are much less known, and their use in therapy of neoplastic and viral diseases is unknown.
In the synthesis of DNA-related oligomers, types of nucleoside dimers are synthesized as part of the overall process. These dimers usually include bases from naturally occurring DNA or RNA sequences. There is much known in the art about nucleoside monophosphate dimers. Many of these compounds have been synthesized and are available commercially. However, these dimers are made from nucleosides containing a sugar moiety in D-configuration.
Reese, C. B., Tetrahedron 34 (1978) 3143 describes the synthesis of fully-protected dinucleoside monophosphates by means of the phosphotriester approach.
Littauer, U. Z., and Soreg, H. (1982) in The Enzymes, Vol. XV, Academic Press, NY, p. 517 is a standard reference which describes the enzymatic synthesis of dinucleotides.
Heikkilo, J., Stridh, S., Oberg, B. and Chattopodhyaya, J., Acta Chem. Scand. B 39 (1985) 657-669, provides an example of the methodology used in the synthesis of a variety of ApG nucleoside phosphate dimers. Included are references and methods for synthesis of 3'.fwdarw.5' phosphates and 2'.fwdarw.5' phosphates by solution phase chemistry.
Gait, M., "Oligonucleotide Synthesis", IRL Press, Ltd., Oxford, England, 1984, is a general reference and a useful overview for oligonucleotide synthesis. The methods are applicable to synthesis of dimers, both by solution phase and solid phase methods. Both phosphitetriester and phosphotriester methods of coupling nucleosides are described. The solid phase method is useful for synthesizing dimers.
Gulyawa, V. and Holy, A., Coll. Czec. Chem. Commun 44 613 (1979), describe the enzymatic synthesis of a series of dimers by reaction of 2',-3' cyclic phosphate donors with ribonucleoside acceptors. The reaction was catalyzed by non-specific RNases. The donors are phosphorylated in the 5'-position, yielding the following compounds: donor nucleoside-(3'.fwdarw.5') acceptor nucleoside. Dimers were made with acceptors, .beta.-L-cytidine, .beta.-L-adenosine, and 9(.alpha.-L-lyxofuranosyl) adenine. Also, a large number of dimers with D-nucleosides in the acceptor 5'-position were made.
Holy, A., Sorm, F., Collect. Czech. Chem. Commun., 34, 3383 (1969), describe an enzymatic synthesis of .beta.-D-guanylyl-(3'.fwdarw.5')-.beta.-L-adenosine and .beta.-D-guanylyl-(3'.fwdarw.5')-.beta.-L-cytidine.
Schirmeister, H. and Pfleiderer, W., Helv. Chim. Acta 77, 10 (1994), describe trimer synthesis and intermediate dimers, all from .beta.-D-nucleosides. They used the phosphoramidite method which gave good yields.
Thus, dimers with L-deoxyribofuranosyl moieties in any position are new, as are dimers with L-ribofuranosyl moieties bonded to the 3'-position of the phosphate internucleotide bond.
Modified nucleoside analogues represent an important class of compounds in the available arsenal 3'of antineoplastic and antiviral drugs. The anticancer agents 5-fluorodeoxyuridine (floxuridine), cytarabine and deoxycoformycin and the antiviral drugs 3'azidodeoxythymidine (AZT), dideoxycytidine (ddC), dideoxyinosine (ddI), acyclovir, 5-iododeoxyuridine (idoxuridine) fludarabine phosphate and vidarabine (adenine arabinoside/ara A) are representative of this class of monomeric nucleoside-derived compounds which are used therapeutically.
More recently, "antisense" oligonucleotide analogues with modified bases and/or phosphodiester backbones have been actively pursued as antiviral and antitumor agents. While no clinically approved drug has yet emerged from this class of compounds, it remains a very active field of research. Recently, antipodal L-sugar-based nucleosides also have found application as potent antiviral agents because they can inhibit viral enzymes without affecting mammalian enzymes, resulting in agents that have selective antiviral activity without concomitant mammalian cytotoxicity.
Most naturally occurring nucleosides have the D-configuration in the sugar moiety. While the chemical properties of L-nucleosides are similar to those of their .beta.-D-enantiomers, they exhibit very different biological profiles in mammalian cells and do not interfere with the transport of normal D-nucleosides. For example, .beta.-L-uridine is not phosphorylated at the 5'-position by human prostate phosphotransferase, which readily phosphorylates the enantiomeric .beta.-D-uridine. Apparently, L-nucleosides are not substrates for normal human cell kinases, but they may be phosphorylated by viral and cancer cell enzymes, allowing their use for the design of selective antiviral and anticancer drugs.
Oligonucleotides based on L-nucleosides have been studied previously. Octamers derived from .alpha.- and .beta.-L-thymidine were found resistant to fungal nucleases and calf spleen phosphodiesterase, which readily degrades the corresponding .beta.-D-oligonucleotide. Fujimory, et al., S. Fujimory, K. Shudo, Y. Hashimoto, J. Am. Chem. Soc., 112, 7436, have shown that enantiomeric poly-.alpha.-DNA recognizes complementary RNA but not complementary DNA. This principle has been used in the design of nuclease-resistant antisense oligonucleotides for potential therapeutic applications.
Thus, L-nucleoside-based compounds have potential as drugs against neoplastic and viral diseases. While L-sugar-derived nucleosides and their oligonucleotides have been widely evaluated for such activities, little is known regarding the biological activities of shorter oligomers such as dimers obtained by L-nucleoside substitution.
This invention comprises novel L-nucleoside-derived therapeutic antitumor and antiviral agents. Novel L-nucleoside-derived dinucleoside monophosphates, based on L-.alpha.-5-fluoro-2'-deoxyuridine showed a remarkably high potency activity profile in in vitro anti-cancer assays, with indications of unique mechanisms of action, including inhibition of telomerase. Therefore, the L-nucleosides can serve as building blocks for new drugs with the special advantage of low toxicity.